Summary of work: We continue to elucidate the mechanisms by which stimulation of DNA synthesis in primary hepatocyte cultures from aged rats is impaired. At least part of the defect appears to be fewer responding cells than in cultures from young rats. The relationship between this reduction in numbers of responding cells and changes in particular signal transduction components and events remains to be determined. However, we have confirmed and extended our observation that EGF stimulation of the MAPK pathway is critical in young cells, but becomes progressively less important during aging as the general response is reduced. Inhibition of stimulated DNA synthesis by the MEK inhibitor, PD 09859, is complete in cells of young rats and decreases to no effect at all in old rats. Thus, alternative pathways of EGF signal transduction seem to be adopted during aging. Two candidates examined recently are the PKC and superoxide pathways. Inhibitors of the former can almost completely block EGF stimulated DNA synthesis in young and old cells, but direct stimulation by phorbol estersis reduced in cells of aged rats. In contract, antioxidants block stimulated DNA synthesis in young cells but not old. Taken together, these preliminary findings suggest that old cells do not preferentially shift to either of these pathways. We have also continued to examine the existing EGF signal transduction pathways in aged hepatocytes. Reductions have been observed in induction of cyclic D1, CDK2 and p70S6 kinase. These findings confirm and extend our earlier results documenting age changes in the MAPK pathway. In order to further confirm the reduction in numbers of responding cells in aged rat cultures we have also begun to establish an in situ hybridization assay for DNA polymerase alpha. When completed it will enable us to document the numbers of cells in young and old rat cultures in which EGF activates this enzyme required for DNA replication. We will continue to attempt to identify the EGF signal transduction pathway preferentially adopted by hepatocytes of old rats. One clue may be the fact that only inhibitors of nuclear events seem to block stimulation in old cells, but inhibitors of both cytoplasmic and nuclear events are effective in young. We will also attempt to further elucidate the causes of the reduction in numbers of responding hepatocytes in aged animals. If in situ hybridization assays for DNA polymerase alpha reveal similar numbers of cells to those incorporating 3H-thymidine, we will examine earlier signal transduction events in individual cells. This approach should allow us to determine the earliest event affected by aging and thus rate limiting for entry into S phase. As always, we will continue to identify various interventions which may both prevent or reverse the functional changes described above. These include insulin supplementation, thymic grafts, caloric restriction and other nutritional manipulations (see also Z01 AG00304-07 and 10).